We have isolated mutant CHO cells which are deficient in binding and uptake of exogenously added lysosomal enzymes, due to alterations in the mannose 6-phosphate receptor. These mutants secrete 50% of their newly synthesized lysosomal enzymes into the growth media, about five times more than is secreted by the parent cells. Lysosomal enzymes secreted by these mutants contain the mannose 6-phosphate recognition marker, thus mislocation of the mutants' enzyme would appear to result from defects in the receptor. Biosynthesis of the recognition marker has been studied in human fibroblasts and CHO cells. Lysosomal enzymes of a mutant deficient in glucosylation of oligosaccharide-lipid were shown to contain complex oligosaccharides instead of the phosphorylated species. The phosphorylated oligossaccharide obtained from wild type CHO and normal human fibroblasts has the composition: N-acetylglucosamine2 mannose6; this structure gives rise to at least three oligosaccharides, including the phosphorylated species, on the mature enzyme.